Replication of DNA is a complex multi-step process essential to cell propagation and survival, which proceeds via the action of multi-protein machines. Understanding the machinery at the replication fork has high impact because it is the most critical site for propagation and maintenance of the genome. While considerable progress has been made in elucidating the mechanisms of DNA replication from studies of bacteria and archae, information on replication in humans is lacking because the protein sequences and structures are not conserved. The long-term goal of our research is to understand the action of the DNA replication machinery in humans. Our research currently focuses on understanding the initiation of the step known as DNA priming. We have shown active loading of human replication protein A (RPA) onto single-stranded DNA (ssDNA) created by the SV40 helicase at the origin of replication and involvement of RPA in the transition to DNA priming. After the DNA is unwound, an initial primer is synthesized on the ssDNA template by primase. The studies proposed here are designed to generate insight into how RPA and primase function together to initiate synthesis of the primer strand.
Aim 1 investigates the structure of RPA in different DNA-bound states using a combination of small angle X-ray and neutron scattering (SAXS, SANS) and NMR spectroscopy.
Aim 2 addresses the role of interactions with RPA in promoting the loading of primase on the template using a combination of biochemical mapping and structural analyses by NMR, modeling, SAXS and SANS. Once primase is loaded on the DNA template, it synthesizes a ~10 nucleotide primer and then transfers the primed template to DNA polymerase a for primer extension. The means by which primase recognizes the template and counts the length of the primer remains a complete mystery.
Aim 3 proposes to elucidate the structural basis for these processes by determining x-ray crystal structures of primase in different DNA bound states. Together, these results will inform the structural basis for the hand-off of ssDNA from RPA to DNA primase and counting of the RNA primer, which are critical steps in the replication of DNA.
Faithful replication and maintenance of our genomes requires the action of complex multi-protein machinery. Defects in components of this machinery lead to mutation and ultimately cancer and other diseases associated with genomic instabilities. Investigating the structure and coordinated action of the complex of proteins involved in initiating replication in humans will provide detailed mechanistic insight into the action of the multi-protein machinery at the replication fork, the most critical site for propagation and maintenance of the genome.
|Feldkamp, Michael D; Mason, Aaron C; Eichman, Brandt F et al. (2014) Structural analysis of replication protein A recruitment of the DNA damage response protein SMARCAL1. Biochemistry 53:3052-61|
|Frank, Andreas O; Vangamudi, Bhavatarini; Feldkamp, Michael D et al. (2014) Discovery of a potent stapled helix peptide that binds to the 70N domain of replication protein A. J Med Chem 57:2455-61|
|Vaithiyalingam, Sivaraja; Arnett, Diana R; Aggarwal, Amit et al. (2014) Insights into eukaryotic primer synthesis from structures of the p48 subunit of human DNA primase. J Mol Biol 426:558-69|
|Hwang, Helen; Kreig, Alex; Calvert, Jacob et al. (2014) Telomeric overhang length determines structural dynamics and accessibility to telomerase and ALT-associated proteins. Structure 22:842-53|
|Frank, Andreas O; Feldkamp, Michael D; Kennedy, J Phillip et al. (2013) Discovery of a potent inhibitor of replication protein a protein-protein interactions using a fragment-linking approach. J Med Chem 56:9242-50|
|Feldkamp, Michael D; Frank, Andreas O; Kennedy, J Phillip et al. (2013) Surface reengineering of RPA70N enables cocrystallization with an inhibitor of the replication protein A interaction motif of ATR interacting protein. Biochemistry 52:6515-24|
|Patrone, James D; Kennedy, J Phillip; Frank, Andreas O et al. (2013) Discovery of Protein-Protein Interaction Inhibitors of Replication Protein A. ACS Med Chem Lett 4:601-605|
|Brosey, Chris A; Yan, Chunli; Tsutakawa, Susan E et al. (2013) A new structural framework for integrating replication protein A into DNA processing machinery. Nucleic Acids Res 41:2313-27|
|Souza-Fagundes, Elaine M; Frank, Andreas O; Feldkamp, Michael D et al. (2012) A high-throughput fluorescence polarization anisotropy assay for the 70N domain of replication protein A. Anal Biochem 421:742-9|
|Brosey, Chris A; Chagot, Marie-Eve; Chazin, Walter J (2012) Preparation of the modular multi-domain protein RPA for study by NMR spectroscopy. Methods Mol Biol 831:181-95|
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